Thermophilic bacteria have received considerable attention as sources of highly active and thermostable enzymes. Interest in DNA polymerases from thermophilic microbes increased with the invention of nucleic acid amplification processes. The use of thermostable enzymes, such as those described in U.S. Pat. No. 4,165,188, to amplify existing nucleic acid sequences in amounts that are large compared to the amount initially present was described U.S. Pat. Nos. 4,683,195 and 4,683,202, which describe the PCR process. These patents are incorporated herein by reference.
The PCR process involves denaturation of a target nucleic acid, hybridization of primers, and synthesis of complementary strands catalyzed by a DNA polymerase. The amplification product of each primer becomes a template for the production of the desired nucleic acid sequence. If the polymerase employed is a thermostable enzyme, polymerase need not be added after every denaturation step, because heat will not destroy the polymerase activity. Thermostable DNA polymerases are not irreversibly inactivated even when heated to 93° C. to 95° C. for brief periods of time, as, for example, in the practice of DNA amplification by PCR. In contrast, at this elevated temperature E. coli DNA Pol I is inactivated.
Archaeal hyperthermophiles, such as Pyrodictium and Methanopyrus species, grow at temperatures up to about 110° C. and are unable to grow below 80 degree. C. (see, Stetter et al., 1990, FEMS Microbiology Reviews 75:1170124, which is incorporated herein by reference). These sulfur reducing, strict anaerobes are isolated from submarine environments. For example, P. abyssi was isolated from a deep sea active “smoker” chimney off Guaymas Mexico at 2,000 meters depth and in 320° C. of venting water (Pley et al., 1991, Systematic and Applied Microbiology 14:245). The hyperthermophile that lives at the highest known temperature, Pyrolobus fumaria, grows in the walls of hydrothermal vents, sometimes called smokers, through which superheated, mineral-rich fluids erupt. Pyrolobus fumaria reproduces best in an environment of about 105° C. and can multiply in temperatures of up to 113° C., but stops growing at temperatures below 90° C.
The more common thermophilic microorganisms have an optimum growth temperature at or about 90° C. and a maximum growth temperature at or about 100° C. These less extreme hyperthermophiles can be grown in culture. For example, a gene encoding DNA polymerase has been cloned and sequenced from Thermococcus litoralis (EP No. 455,430). However, culture of the extreme hyperthermophilic microorganisms is made difficult by their inability to grow on agar solidified media. For example, individual cells of the Pyrodictium species are extremely fragile, and the organisms grow as fibrous networks, clogging the steel parts of conventional fermentation apparatus. Thus, standard bacterial fermentation techniques are extremely difficult for culturing Pyrodictium. (See Staley, J. T. et al. eds., Bergey's Manual of Systematic Bacteriology, 1989, Williams and Wilkins, Baltimore, which is incorporated herein by reference.) These and other difficulties may preclude laboratory culture for preparing large amounts of purified nucleic acid polymerase enzymes for characterization and amino acid sequence analysis.
There is a desire in the art to produce thermostable DNA polymerases having enhanced thermostability that may be used to improve the PCR process and to improve the results obtained when using a thermostable DNA polymerase in other recombinant techniques such as DNA sequencing, nick-translation, and reverse transcription.